Use of angelicin and of its structural analogues for the treatment of thalassemia

ABSTRACT

The use of angelicin and its structural analogues for the preparation of a medicament for the therapeutic treatment of beta-thalassaemia is described. A structural analogue which is particularly preferred for this purpose is bergapten.

This is a National Stage entry of International ApplicationPCT/IB2003/003462, with an international filing date of Jul. 30, 2003,which was published under PCT Article 21(2) as WO 2004/012729 A1, andthe complete disclosure of which is incorporated into this applicationby reference.

The present invention relates to a novel therapeutic use of angelicinand its structural analogues as molecules capable of inducing erythroidcell differentiation and of increasing the production of gamma-globinmRNA.

The existence of substances capable of inducing the expression of thegamma-globin gene and the biosynthesis of foetal haemoglobin (HbF) inadult subjects has been known for some time (1). In the majority ofcases, those substances are also capable of activating or potentiatingthe transcription of genes for embryonic and foetal globins inexperimental model systems.

Recently, for example, numerous DNA-binding molecules have beendescribed that have the capacity to bring about an increase in theexpression of genes for gamma-globin (2). Among these there may bementioned cisplatin and analogues of cisplatin, mithramycin,chromomycin, and tallimustine (3). Such molecules are efficientmodulators of the expression of gamma-globin genes.

In human beings, activation of the transcription of genes forgamma-globins in adult subjects leads to the production of foetalhaemoglobin mimicking the phenotype HPFH (High Persistence of FoetalHaemoglobin) which confers a favourable clinical picture on patientssuffering from beta-thalassaemia also in homozygotic form (4). A therapyproviding for the use of molecules having such activity for thetreatment of patients suffering from beta-thalassaemia could thereforemake those subjects less dependent on transfusion therapy (5).

The present invention is based on the need for novel modifiers of thetranscription process which can be used in the treatment ofbeta-thalassaemia and which have a high level of induction of theexpression of gamma-globin genes and at the same time a lower cytotoxiceffect than reference drugs.

We have surprisingly found that angelicin—an isopsoralen derivativewhich has photochemotherapic activity—as well as its structuralanalogues, possesses such activity. In particular, it has been foundthat angelicin and its structural analogues are capable of potentiatingthe expression of the gene for human gamma-globin.

This activity is unexpected in the light of the known therapeutic usesof angelicin and of its structural analogues (6-18).

Angelicin has in fact been proposed in the literature for the treatmentof psoriasis (12, 13), as an antiproliferative agent (14, 15, 16), as anantifungal agent (8), and as an anti-inflammatory agent (17).

The structural formula of angelicin is as follows:

The chemical synthesis of angelicin and its structural analogues hasbeen described in the literature (see, for example, references 6-11).

Structural analogues of angelicin are, for example, linear and angularcoumarins, optionally substituted, heteroanalogues of angelicin,thiopyrano-benzofurans, acylangelicins, alkylangelicins,alkoxycarbamoylangelicins, psoralens and isopsoralens, optionallysubstituted. A specific example is the linear furanocoumarin analoguebergapten (5-methoxypsoralen) which is currently used in PUVA (Psoralensplus UVA radiation) therapy for the treatment of psoriasis.

The structural formula of bergapten is as follows:

By virtue of their capacity to potentiate the expression of the gene forhuman gamma-globin, angelicin and its structural analogues canadvantageously be used for the therapeutic treatment of patientssuffering from beta-thalassaemia.

A first subject of the present invention is therefore the use ofangelicin or a structural analogue thereof for the preparation of amedicament for the therapeutic treatment of beta-thalassaemia.

The structural analogue is preferably selected from the group consistingof linear and angular coumarins, optionally substituted, heteroanaloguesof angelicin, thiopyrano-benzofurans, acylangelicins, alkylangelicins,alkoxycarbamoylangelicins, psoralens, and isopsoralens, optionallysubstituted.

A particularly preferred structural analogue is bergapten.

Moreover, as has been recently described (18, 19), a combined treatmentwith various modifiers of the transcription process permits a furtherincrease in the expression of genes for gamma-globin.

Therefore, a second subject of the present invention is the use ofangelicin or a structural analogue thereof in combination with at leastone further modifier of the transcription process for the preparation ofa medicament for the treatment of beta-thalassaemia.

According to a preferred embodiment, the further modifier of thetranscription process is selected from the group consisting of cytosinearabinoside, retinoic acid, plicamycin, hydroxyurea, guanine, guanosinetriphosphate (GTP), guanosine diphosphate (GDP) and guanosinemonophosphate (GMP). Of these, cytosine arabinoside and retinoic acidare more preferred.

The activity of angelicin as an inducer of erythroid celldifferentiation and the production of gamma-globin mRNA has beenevaluated in human cell cultures.

The results of this study are illustrated in the following Examples. Thedata given in the Examples indicate that the activity of angelicin isgreater than that of hydroxyurea which is a reference drug for theinduction of foetal haemoglobin (HbF). It has also been ascertained thatthe cytotoxic effect which may be encountered is much lower than that ofhydroxyurea.

The Examples which follow are provided for the purposes of illustrationand are not intended to limit the scope of the invention in any way.

EXAMPLE 1

The biological activity of angelicin was evaluated by examining thecapacity of that compound to modulate the expression of genes forgamma-globin in the human cell line K562, which is capable ofdifferentiating in the erythroid manner by expressing genes forgamma-globin if subjected to treatment with modifiers of the biologicalresponse that are suitable for the purpose (3, 18, 19).

The level of differentiation was evaluated by analyzing the positivereaction of the cells to benzidine (3). The production of haemoglobinwas evaluated by electrophoresis on cellulose acetate and by colouringthe gel with a solution based on benzidine/H₂O₂ (3). The expression ofthe genes encoding for gamma-globin was evaluated by quantitative RT-PCR(reverse transcriptase PCR) (3).

Some of the data obtained are given in Table 1. As will be readilyappreciated, angelicin (400 μM) is capable of bringing about an increasein the percentage of K562 cells that react positively to benzidine(55-60% of the cells treated, compared with 3-4% of the control K562cells). The chief haemoglobin produced by K562 cells treated withangelicin was Hb Portland (alpha2gamma2). The data obtained byquantitative RT-PCR demonstrate that this effect on differentiation isassociated with an increase in the intracytoplasmic accumulation of mRNAfor gamma-globin. These evaluations were carried out after inductionwith 400 μM angelicin for 6 days.

Bergapten also showed the capability to bring about differentiation(measured as increase in cells that react positively to benzidine),although this capability was associated with a lesser increase in theaccumulation of mRNA for globin.

The induction of erythroid differentiation in cells treated withangelicin is very similar to that obtained with cytosine arabinoside,which is one of the most effective known inducers (18, 19). However, theactivity of angelicin on the increase in production of mRNA forgammma-globin is significantly greater than that of cytosinearabinoside.

TABLE 1 ^((a))Erythroid Concentration differentiation ^((b))Gamma-globinCompound (μM) (%) mRNA — — 3-4 1 cytosine arabinoside 1 75-80 3.24bergapten 200 50-60 3.48 angelicin 400 55-60 44.94 ^((a))Erythroiddifferentiation = percentage of K562 cells that react positively tobenzidine (mean ± SD of six experiments). The concentrations indicatedare those that are optimal for each molecule in order to activate theerythroid differentiation. Increasing these concentrations brings abouta reduction in the effect on the differentiation parameters incombination with a cytotoxic activity of the molecules themselves.^((b))The accumulation of RNA for gamma-globin is given in the Table asan increase compared with that of untreated control K562 cells. Thetechnique used was that of quantitative RT-PCR (22, 23) using thefollowing primer and probe oligonucleotides: gamma-globin forwardprimer, 5′-TGG CAA GAA GGT GCT GAC TTC-3′ (SEQ ID NO: 1); gamma-globinreverse primer, 5′-TCA CTC AGC TGG GCA AAG G-3′ (SEQ ID NO: 2);gamma-globin probe 5′-FAM-TGG GAG ATG CCA TAA AGC ACC TGG-TAMRA-3′ (FAM= 6-carboxy fluorescein, TAMRA =6-carboxy-N,N,N′,N′-tetramethyl-rhodamine) (SEQ ID NO: 3). The analyseswere carried out on material extracted from cells treated for 6 days inthe conditions indicated.

EXAMPLE 2

In order to check whether angelicin was also capable of stimulating theproduction of mRNA for gamma-globin in human erythroid precursorsisolated from peripheral blood, the technique published by Fibach et al.(20, 21) was used. This technique provides for two stages: in the firststage, the cells isolated from peripheral blood of a subject who ishealthy or suffering from a haemopoietic pathology, such as sickle cellanaemia or beta-thalassaemia, are sown in a culture medium to which 10%of conditioned medium derived from the vesicle carcinoma cell line 5637has been added. The second stage consists in cultivating the isolatedcells in a suitable culture medium, supplemented by the erythropoietinhormone, 30% bovine foetal serum, 2-mercaptoethanol, albumin, glutamineand desamethasone in order to permit the proliferation and maturing ofstem cells of the erythroid type. In this stage the cells can be treatedwith potential HbF inducers. For example, with this system it wasdemonstrated that hydroxyurea, an inhibitor of DNA synthesis currentlyused in the experimental therapy of beta-thalassaemia, is capable ofbringing about the production of HbF.

The results obtained by this technique demonstrated an increase in theproduction of mRNA for gamma-globin in cells treated with angelicincompared with the same untreated cells (20.53 times). This increase isgreater than that which may be encountered using hydroxyurea (Table 2).

TABLE 2 Compound Concentration (μM) ^((a))Gamma-globin mRNA — — 1angelicin 500 20.53 hydroxyurea 120 6.96 ^((a))The accumulation of RNAfor gamma-globin is given in the Table as an increase compared with thatof untreated control erythroid precursors. The technique used was thatof quantitative RT-PCR (22, 23) using the primer and probeoligonucleotides described in Table 1. The hydroxyurea was used at aconcentration of 120 μM since this molecule has a high cytotoxicactivity at concentrations comparable with those used for angelicin.This highly cytotoxic activity starts to be encountered atconcentrations greater than 250-300 μM.

BIBLIOGRAPHY

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1. A method for treating beta-thalassaemia, comprising administering toa subject in need of treatment an effective amount of angelicin,represented by formula (1):

or a structural analogue thereof, wherein the structural analogue isselected from the group consisting of an acylangelicin, analkylangelicin, an alkoxycarbamoylangelicin and bergapten.
 2. The methodaccording to claim 1, wherein the angelicin or structural analogue is incombination with at least one further modifier of the transcriptionprocess, wherein the further modifier of the transcription process isselected from the group consisting of cytosine arabinoside, retinoicacid, plicamycin, mithramycin, hydroxyurea, guanine, guanosinetriphosphate (GTP), guanosine diphosphate (GDOP) and guanosinemonophosphate (GMP).